A.P. Biology Chapters 16, 17, 18
Molecular Basis of Reproduction; Biology of the Gene, viral life cycle
Deoxyribonucleic acid or DNA = genetic material
Inheritance is based on the replication and transmission of DNA from parent to offspring.
Search for genetic material
By 1940’s, scientists knew chromosomes carry hereditary material and consists of DNA and protein.
Most thought protein was the genetic material.
Frederick Griffith--mice
Performed experiments which provided evidence that genetic material is a specific molecule
Two types of pneumonia-smooth (S) and rough (R)
Smooth cells encapsulated with a polysaccharide coat and rough cells are not encapsulated.
Alternative phenotypes (S and R) are inherited traits. [i.e. r produces more r]
Transformation-the absorption and incorporation of external genetic material by a cell
Griffith’s experiments hinted that protein is not the genetic material
In 1944 Avery, McCarty and MacLeod discovered the transforming agent had to be DNA--continuation of
Griffiths’ work with the heat killed S and R cells
Bacteriophage – a type of virus that infects bacteria
Hershey and Chase discovered DNA is the genetic material of a phage known as T2--- in a blender
experiment.
A T2 bacteriophage can quickly reprogram an E. coli cell to produce more T2 phages and release
the viruses when the cell lyses (bursts)
Radioactive tagging-Virus protein and DNA was tagged with different radioactive isotopes
Concluded DNA is injected into the host cell, causing cells to produce viral DNA and proteins.
Injected material was DNA provides evidence nucleic acids, rather than proteins, are the hereditary
material.
Additional Evidence that nucleic acid is the genetic material
*A eukaryotic cell doubles its DNA prior to mitosis
*During mitosis, DNA is equally divided between two daughter cells
*An organism’s diploid cells have twice the DNA as its haploid gametes
*DNA composition is species- specific
*Regularity in base ratios
number of adenine (A) residues equaled the number of thymine (T), guanines (G) equaled cytosine (C)
Discovery of Double Helix
Three dimensional structure of DNA was found by X-ray crystallography-- x-ray photo of DNA.
Deduced that:
DNA is a helix with a uniform width of 2nm. Width suggests it has two strands
Proposed a specific pairing between base pairs
Information on one strand complements that along the other
The sequence of bases can be highly variable which makes it suitable for coding genetic
information
Hydrogen bonds between paired bases are weak bonds, yet stabilize the DNA molecule.
DNA Replication
Template replication
Genes on the original DNA strand are copied by a specific pairing of complementary bases, creating a
complimentary DNA strand
Complementary strand can then function as a template to produce a copy of the original strand
1) Two DNA strands separate
2) Each strand is a template
3) Nucleotides line up singly along the template strand in accordance to base-pairing rules (A-T, GC)
4) Enzymes link nucleotides together at their sugar phosphate groups
Semi conservative model-Meselson Stahl experiment
When a double helix replicates, each of the two daughter molecules will have one old or conserved
strand from the parent molecule and one new strand. “heavy” and “light” nucleotides
Closer Examination of DNA Replication
DNA replication is:
Complex - helical molecule untwists while copying two anti parallel strands simultaneously
Extremely rapid - up to 500 nucleotides are added per second.
Accurate-only one in a billion nucleotides is incorrectly paired
Origins of Replication-special sites having a specific sequence of nucleotides where DNA
replication begins
Double helix opens and replication forks spread in both directions, creating a replication bubble
Thousands of replication bubbles merge, forming two continuous DNA molecules
Elongating a New DNA Strand:
Strand separation- two types of proteins involved in separation of parental DNA
1) helicases-catalyze unwinding of parental DNA
2) single-strand binding proteins- keep the separated strands apart and stabilize unwound DNA
Synthesis of the New DNA Strands
DNA polymerase- enzyme that catalyze new DNA strand synthesis
New nucleotides align themselves along the templates of the old DNA strands according to basepairing rules
Strands grow in 5’-3’ direction, since nucleotides are added only to the 3’ end
Nucleoside triphosphates- nucleotides with three phosphates linked to the 5’ carbon; its hydrolysis
provides energy to synthesize new DNA
Leading strand- DNA strand which is synthesized as a single unit in the 5’-3’ direction
Lagging strand- DNA strand which is synthesized discontinuously against the overall direction of
replication lagging strand is produced in short fragments, Okazaki fragments, and linked by DNA
ligase
helicases unwind and separate ‘old’ DNA
single strand binding proteins stabilize the single stranded DNA
add RNA primer--attracts DNA polymerase
add DNA polymerase--adds complimentary bases to old DNA
leading strand continuous
lagging strand fragments joined by ligase
Priming-primers start the addition of new nucleotides
Primer-short RNA segment complementary to a DNA segment that is necessary to begin DNA
replication
Proofreading
Pairing errors- one in a thousand; errors in complete DNA molecules- one in one billion
DNA Repair
Accidental changes in DNA can result from exposure to chemicals, radioactivity, X-rays and ultraviolet
light
Cells monitor accidental changes and repair most
More than 50 enzymes can repair damage by:
*directly reversing the change
*excising the damaged segment, nuclease enzyme. by one repair enzyme and filling the gap, DNA
polymerase. with the undamaged strand then sealing the gap, ligase
Instructions in DNA direct protein synthesis; protein is the link between genotype and phenotype.
Genes specify proteins
dictate phenotypes through enzymes that catalyze reactions
Genes control metabolism
genes direct production of specific enzymes
cells synthesize/degrade compounds via metabolic pathways; each step is catalyzed by a specific
enzyme
ex. Drosophila, eye color; Neurospora, bread mold
One gene-one polypeptide
Beadle and Tatum experiment
mold neurospora x-ray induced mutations
3 types interrupting a sequence of chemical reactions at a different
location for
each mutation
Protein Synthesis
RNA transcribes genetic instructions from DNA and translates that message into a protein
RNA, like DNA, is a nucleic acid or polymer of nucleotides
RNA-sugar is ribose, versus deoxyribose
uracil instead of thymine
location
codons--three nitrogen bases in a row that dictate which type of amino acid is to be place in each
within a protein.
for ex. a sequence of AUG nucleotides would cause the placement of methionine at that location
the sequence AUG was created when DNA was copied into RNA a process called transcription.
transcription [may be transcribed by many RNA polymerases at a time = large production of that protein]
INITIATION
RNA polymerase attaches to the DNA at locations called promoters which are located at the
beginning of
each gene.
in eukaryotes a region rich in A & T’S called a tata box is also needed to bind RNA polymerase to
the DNA
ELONGATION
The RNA polymerase untwists one turn of DNA and opens up the DNA strand
RNA polymerase adds RNA nucleotides to the exposed DNA strand and,
joins the RNA nucleotides into a long molecule of RNA.
the growing RNA molecule separates from the DNA strand as it grows which allows the DNA to
rejoin.
TERMINATION
when the RNA polymerase reaches a DNA series AATAAA it breaks free from the DNA
in bacteria the RNA is used to form proteins
in eukaryotes there is non useful information [introns] that must be cut out before the remaining RNA can be
used [expressed--exons]
RNA PROCESSING
RNA splicing, removes introns and joins exons. spliceosome, large complex of chemicals that
change RNA
to mRNA.
introns help separate genes thus increasing the chance for recombination during meiosis
introns/exons may not be the same for two different cell types. thus, exons in one cell might be
introns in
another--so a gene could code for more than one protein depending upon how it
was processed.
a protective cap and tail are added to the mRNA to slow the attack of hydrolytic enzymes and may
be
needed to attach the mRNA to the ribosome
TRANSLATION
location of translation--the ribosome
ribosomes are composed of two subunits [large and small]
constructed in the nucleus
prokaryotic ribosomes different than eukaryotic cell ribosomes [some drug therapies are
based on
this difference--tetracycline and streptomycin interfere with
bacterial ribosomes]
mRNA binding site, P site-holds the tRNA carrying the peptide chain, A site holds the
tRNA carrying
the next AA to be added
building a polypeptide
occurs in three stages
1. initiation
mRNA joins with a special tRNA binds to the mRNA first codon AUG,
carries methionine
the ribosomal subunit then binds with the mRNA
the large ribosomal subunit binds to the small one = functional ribisome
2. elongation
a tRNA with a matching anti codon [matches the mRNA codon] enters
this tRNA
the A site.
enzymes catalyze a bond between the two A.A. [peptide bond]
the tRNA in the P site is released.
the tRNA at the A site is translocated to the A site
about 30 milliseconds per step
3. termination
eventually a termination codon will reach the ribosome.
a protein release factor binds to the codon
hydrolyzes the bond between the polypeptide and the last
tRNA
frees the polypeptide and the tRNA from the ribosome
ribosome separates
polyribosome--many ribosomes on one mRNA at one time = many proteins in production
free ribosomes --protein for use in cytosol
bound ribosomes--proteins to be used within cellular membranes or secretory proteins to be exported
mutations
point mutations
substitutions
base pair substitution
results in change of an A-T or C-G pair-often little or no effect
missense mutation a base pair mutation that codes for a different AA at that location
nonsense mutation a base pair mutation that creates a termination code in a location it
does not belong or removes a termination code.
insertions/deletions-usually a greater negative effect
base pair insertion
base pair deletion
frame shift mutation-a shift in the reading frame so all triplet codes are changed
if an insertion or deletion is a multiple of three the effect is not a frame shift
mutagens--chemical or physical agents that interact with DNA to cause mutations . radiation
Scientists discovered the roll of DNA in heredity from studying the viruses and bacteria
Viruses-first studied caused tobacco mosaic disease
1) nucleic acid in protein coat
knowledge used for: how disease is caused by viruses and bacteria
gene manipulation
2) reproduce only within living cells
genomes- The entire set of genes in a species can be either:
DNA-- double or single stranded
RNA-- double or single stranded
protein coat--Capsid
rod shape
polyhedral
complex
ex. tobacco mosaic virus or adenoviruses
Envelope-(some viruses)membrane covers the capsid
source : host cell
helps infect the host
Bacteriaophage (Phage)--most complex capsids
bacterial virus
Replication of Viruses occurs only in a host cell
viruses are host specific -host range*a host cell has surface receptors
*the virus has external proteins to match [hook to] the host
1) attachment
2) infection of host
nucleic acid injected into host
3)viral genome replication
DNA ---> DNA
host enzymes
RNA ---> RNA
RNA replicase
RNA ---> DNA------> RNA
reverse transcriptase
3a) capsid production
host provides : enzymes, ribosomes, +RNA, A.A., and ATP to produce new viral proteins and
nucleic acid
which spontaneously assemble
4)exit host
There are 2 cycles viruses may follow:
1)lyctic cycle
2)lysogenic cycle (SOME viruses)
Lyctic Cycle
20 - 30 minutes 1 ---> 100’s of viruses at [37 centigrade]
lyses or kills the host
1) attatchment
2) injection DNA
3) enzymes destroy host DNA
4) phage genome directs DNA & protein synthesis
5) host lysis
*lysosomes digest cell wall
*osmotic swelling and rupture
*release of new viruses
Bacteria defenses
1) mutate receptor site
2) restriction enzymes destroy foreign DNA
viruses and bacteria always changing --Coevolution-Lysogenic cycle [2 possible modes of reproduction]
*viral DNA is placed into the host cell DNA
*host is not immediately destroyed
1) virus attatchment
2) injection
3) DNA (viral) forms a circle and begins a lytic or lysogenic cycle
4) lysogenic cycle-enzymes cut host DNA making “sticky ends”
5) DNA (viral) is inserted [recombines] with host DNA. This inserted viral DNA is called a Prophage
prophage
*remains inactive for a time
*replicated in host DNA during bacterial reproduction
The prophage may leave the bacterial chromosome and enter a lytic cycle
Enviromenental factors may help cause this
ex.
herpes environmental factors (sunlight) stress
if a prophage gene is expressed by a bacteria, the bacteria may develop a new
phenotype and cause disease [diptheria, botulism, scarlet fever]
Animal Viruses
1. viruses with envelopes
1.attatchment
2.entry
*envelope fuses with plasma membrane
*entire vurus enters
3.uncoating
4.viral RNA & protein synthesis
5.assembly and release
Herpes virus
double stranded DNA
*envelope from nuclear membrane
*reproduce in host nucleus
<:
*may integrate into host’s genome, which is called a provirus
*stress (physical, emotional) may cause provirus to begin productive cycle
2. RNA viruses
one type is a retrovirus
reverse transcriptase
transcribes DNA from viral RNA
ex. HIV--AIDS
1) attatchment and entry
2) uncoating single RNA
3) reverse transcription RNA molecule is used to produce DNA
4) integration of viral DNA into host DNA provirus, remains indefinately
expression of proviral genes may:
cause expression of oncogenes transform cell into a cancerous state
---or--produce and release new virons.
prion--short for proteinaceous infectious particle that lacks nucleic acid (by analogy to virion) — is
a type of infectious agent made only of protein. Prions are believed to infect and propagate by
refolding abnormally into a structure which is able to convert normal molecules of the protein into
the abnormally structured form, and they are generally quite resistant to denaturation by
protease, heat, radiation, and formalin treatments[
Creutzfeldt-Jakob disease
DNA TRANSFER
Transformation, Transduction, Conjugation/two bacteria join and a plasmid is transferred or
recombination-a piece of DNA is copied and transferred]
Transformation
bacteria assimilated genetic material from the surroundings—mouse experiment
is either destroyed, or assimilated into the DNA
many bacteria have surface proteins that recognized and import DNA
*e. coli incubated in medium with high Ca+2 stimulates DNA uptake
*used by biotechnology industry to introduce foreign genes [e.g. insulin, HGH]
Transduction
gene transfer from one bacteria to another via bacteriophage
*accidental packaging host DNA into viral capsid
*DNA injected into another bacteria transferring it.
Conjugation
two bacteria join and a plasmid is transferred or recombination-a piece of DNA is copied and
transferred]
plasmids are loops of DNA that can be copied and transferred
R plasmids serious problems in medicine in that they carry genes which destroy
antibiotics.
Control of gene expression in prokaryotes
controlling metabolism
1) regulate enzyme activity
feedback inhibition <:
end product shuts off production
2) regulate gene expression
product inhibits m RNA production
Operons see text and lecture